Pulmonary fibrosis is a common disorder without effective pharmacological treatment. The metabolism of collagen, the main component of fibrotic tissue, is a dynamic process with the balance between collagen production and degradation determining tissue architecture. The pathways responsible for removal of collagen that has accumulated in areas of fibrosis are poorly understood. Whether therapies aimed at increasing collagen uptake will benefit patients with established fibrotic disease is unknown. We have recently described a novel pathway of collagen turnover that regulates the severity of tissue fibrosis. We have shown that the glycoprotein Mfge8 binds collagen that has accumulated in the extracellular matrix in areas of fibrosis. Mfge8-bound collagen is then taken up by macrophages for intracellular degradation. Mice deficient in Mfge8 develop an exaggerated fibrotic response after lung injury due to an in vivo defect in collagen degradation. We plan to build upon these findings by identifying other novel pathways by which collagen is taken up by cells. We have designed a Drosophila-based high-throughput screen of collagen uptake. In these studies we plan to identify novel mediators of collagen turnover through this screen and verify the function of human and orthologs of these genes. We will accomplish these goals through two specific aims. In the first aim, we will use a flow cytometry-based high-throughput screen of Drosophila S2 cell collagen uptake using a double stranded RNA library to identify candidate molecules involved in collagen turnover. The library created at UCSF covers Drosophila genes that have human and/or murine orthologs. Drosophila S2 cells are macrophage-like phagocytes that are highly susceptible to gene silencing with double-stranded RNA. We will subsequently rescreen candidate genes with both flow cytometry-based and fluorescent microscopy-based assays of collagen uptake. Candidate genes that remain after the secondary screen will be the focus of our second aim. In these studies we will confirm the function of human and murine orthologs of candidate molecules in collagen turnover. We will select the most compelling candidate genes identified through our Drosophila screen and evaluate their role in collagen turnover in both human and murine cell lines and primary cells. Our approach will involve both silencing candidate genes using shRNA and increasing candidate genes protein levels by adding exogenous recombinant protein. We will then test the effect of these interventions on collagen uptake and turnover by macrophages and fibroblasts. These studies have the potential to identify novel mediators of collagen turnover. The importance of a better understanding of the pathways responsible for collagen removal from the extracellular matrix is highlighted by the fact that no available therapies are efficacious in the treatment of patients with established fibrotic disease.
Pulmonary fibrosis is a common disorder without effective pharmacological treatment options.
Research aim ed at discovering pathways that remove fibrotic scar from the lung will provide new therapeutic options. The goals of this project are to identify and characterize the normal pathways that mediate scar remodeling with the goal of discovering new therapeutic targets for the treatment of pulmonary fibrosis.
| Lee, Ting-Hein; McKleroy, William; Khalifeh-Soltani, Amin et al. (2014) Functional genomic screen identifies novel mediators of collagen uptake. Mol Biol Cell 25:583-93 |
